The present invention is in the field of animal health, and is directed to vaccines that protect swine against Actinobacillus pleuropneumoniae. More particularly, the present invention is directed to novel antigenic proteins shared by multiple serotypes of A. pleuropneumoniae, DNA molecules encoding the proteins, vaccines against APP comprising the proteins, and diagnostic reagents.
A. pleuropneumoniae (hereinafter referred to as xe2x80x9cAPPxe2x80x9d) is a Gram negative coccobacillus recognized as one of the most important swine pneumonic pathogens (Shope, R. E., 1964, J. Exp. Med. 119:357-368; Sebunya, T. N. K. and Saunders, J. R., 1983, J. Am. Vet. Med. Assoc. 182:1331-1337). Twelve different serotypes have been recognized which vary in geographic distribution (Sebunya, T. N. K. and Saunders, J. R., 1983, above; Nielsen, R., 1985, Proc. Am. Assoc. Swine Pract. 18-22; Nielsen, R., 1986, Acta. Vet. Scand. 27:453-455). Immune responses to vaccination against APP have been mainly serotype-specific, suggesting that vaccine-induced immunity is directed to serotype-specific capsular antigens (MacInnes, J. I.). and Rosendal, S., 1988, Can. Vet. J. 29:572-574; Fedorka-Cray, P. J., et al., 1994, Comp. Cont. Educ. Pract. Vet. 16:117-125; Nielsen, R., 1979, Nord. Vet. Med. 31:407-413; Rosendal, S., et al., 1986, Vet. Microbiol. 12:229-240).
In contrast, natural immunity to any one serotype seems to confer significant protection from disease caused by other serotypes, suggesting that natural exposure induces cross-reactive immunity to shared antigens (Sebunya, T. N. K. and Saunders, J. R., 1983, above; Macinnes, J. I. and Rosendal, S., 1988, above; Fedorka-Cray, P. J., et al., 1994, above; Nielsen, R., 1979, above; and Rosendal, S., et al., 1986, above).
Virulence factors that might contribute to cross-protection have been proposed as possible vaccine candidates, including exotoxins (Apx) (Nakai, T., et al., 1983, Am. J. Vet. Res. 30 44:344-347; Frey, J., et al., 1993, J. Gen. Microbiol. 139:1723-1728; Fedorka-Cray, P. J., et al., 1993, Vet. Microbiol. 37:85-100); capsular antigens (Rosendal, S., et al., 1986, above); outer-membrane proteins (OMP) (Denee, H. and Potter, A., 1989, Infect. Immune 57:798-804; Niven, D. F., et al., 1989, Mol. Microbiol. 3:1083-1089; Gonzalez, G., et al., 1990, Mol. Microbiol. 4:1173-1179; Gerlach, G. F., et al., 1992, Infect. Immun. 60:3253-3261); and lipopolysaccharides (LPS) (Fenwick, B. W. and Osborn, B., 1986, Infect. Immun. 54:575-582). However, the patterns of cross-reactivity/cross-protection induced by such components do not cover all twelve APP serotypes. In addition, immunization with isolated individual components or combinations of individual components from APP have so far failed to confer protection from challenge with some heterologous serotypes (unpublished observations). Thus, it can be postulated that the cross-protective responses induced by natural infection are limited to specific serotype clusters.
Alternatively, it is possible that some of the antigens responsible for the cross-protection observed after natural infection have not yet been identified. Most studies regarding APP antigens have focused on the characterization of immunodominant antigens detected in convalescent serum using antibodies. Such an approach does not allow the identification of possible differences between the antibody specificities represented during primary versus secondary responses, nor the identification of dominant specificities at the infection site that are likely to be responsible for protection upon secondary encounter with the pathogen.
It is generally accepted that lymphocytes are educated during primary infections so that when there is secondary exposure to a pathogen the host is better able to prevent disease (MacKay, C. R., 1993, Adv. Immunol. 53:217-240). Memory cells responsible for this activity (antigen-experienced T and B lymphocytes) persist for long periods of time, and are capable of reactivation following an appropriate subsequent encounter with the antigen. In contrast to naive cells, they generally show a faster response time, specialized tissue localization, and more effective antigen recognition and effector functions (MacKay, C. R., 1993, above; Linton, P. and Klinman, N. R., 1992, Sem. Immun. 4:3-9; Meeusen, E. N. T., et al., 1991, Eur. J. Immunol. 21:2269-2272).
During the generation of a secondary response, the frequency of precursor cells capable of responding to the particular antigen is higher than that present during the primary response. Trafficking patterns of memory cell subsets following secondary responses are also different from those of naive cells. Naive cells migrate relatively homogeneously to secondary lymphoid tissues, but they home poorly to non-lymphoid tissues. By contrast, memory cells display heterogeneous trafficking and, in some instances, migration has been shown to be restricted to certain secondary lymphoid tissues and non-lymphoid sites (MacKay, C. R., 1993, above; Gray, D., 1993, Ann. Rev. Immunol. 11:49-77; Picker, L. S., et al., 1993, J. Immunol. 150:1122-1136). Studies in both rodents and sheep have indicated that lymphocytes from the gut preferentially migrate back to the gut, whereas cells draining from the skin or from lymph nodes preferentially migrate back to the skin or lymph nodes (Gray, D., 1993, above; Picker, L. S., et al., 1993, above). Thus, upon secondary encounter with a pathogen, specific effector cells for cell-mediated immunity and antibody secretion can home to infection sites and local lymph nodes more effectively (Meeusen, E. N. T., et al., 1991, above). As a result, infiltrating lymphocytes will rapidly proliferate and their specificities will predominate during early stages of re-infection.
Recovery of local B cells from tissues and draining lymph nodes early after re-infection has allowed some researchers to obtain antibodies with a narrower specificity range (Meeusen, E. N. T. and Brandon, M., 1994, J. Immunol. Meth. 172:71-76). Such antibodies have been successfully used to identify potential protective antigens to several pathogens (Meeusen, E. N. T. and Brandon, M., 1994, above; Meeusen, E. N. T. and Brandon, M., 1994, Eur. J. Immunol. 24:469-474; Bowles, V. M., et al., 1995, Immunol. 84:669-674). The invention disclosed herein below is based on a modification of this approach, in which antibody-secreting cell (ASC) probes were recovered that were associated with local memory responses elicited after homologous and heterologous APP challenge. Antibodies obtained from bronchial lymph node (BLN) cultures after heterologous challenge recognized four previously unrecognized proteins present in all twelve APP serotypes. Partial amino acid sequences for each protein were obtained and used to generate PCR primers that allowed the identification of five novel APP proteins and polynucleotide molecules that encode them.
The present invention provides five novel, low molecular weight proteins isolated from APP, which are designated herein, respectively, as xe2x80x9cOmp20,xe2x80x9d xe2x80x9cOmpW,xe2x80x9d xe2x80x9cOmp27,xe2x80x9d xe2x80x9cOmpA1,xe2x80x9d and xe2x80x9cOmpA2xe2x80x9d. These xe2x80x9cAPP proteinsxe2x80x9d and the polynucleotide molecules that encode them are useful either as antigenic components in a vaccine to protect swine against APP, or as diagnostic reagents to identify swine that are, or have been, infected with APP, or that have been vaccinated with a vaccine of the present invention.
The amino acid sequence of Omp20 is encoded by the Omp20-encoding ORF of plasmid pER416 which is present in host cells of strain Pz416 (ATCC 98926), and its deduced amino acid sequence is presented as SEQ ID NO:2, which comprises a signal sequence from amino acid residues 1 to 19. The amino acid sequence of OmpW is encoded by the OmpW-encoding ORF of plasmid pER418 which is present in host cells of strain Pz418 (ATCC 98928), and its deduced amino acid sequence is presented as SEQ ID NO:4, which comprises a signal sequence from amino acid residues 1 to 21. The amino acid sequence of Omp27 is encoded by the Omp27-encoding ORF of plasmid pER417 which is present in host cells of strain Pz417 (ATCC 98927), and its deduced amino acid sequence is presented as SEQ ID NO:6, which comprises a signal sequence from amino acid residues 1 to 27. The amino acid sequence of OmpA1 is encoded by the OmpA1-encoding ORF of plasmid pER419 which is present in host cells of strain Pz419 (ATCC 98929), and its deduced amino acid sequence is presented as SEQ ID NO:8, which comprises a signal sequence from amino acid residues 1 to 19. The amino acid sequence of OmpA2 is encoded by the OmpA2-encoding ORF of plasmid pER420 which is present in host cells of strain Pz420 (ATCC 98930), and its deduced amino acid sequence is presented as SEQ ID NO:10, which comprises a signal sequence from amino acid residues 1 to 19. Each of these APP proteins, in substantially purified form, is provided by the present invention.
The present invention further provides substantially purified polypeptides that are homologous to any of the aforementioned APP proteins of the present invention. The present invention further provides peptide fragments of any of the APP proteins or homologous polypeptides of the present invention. The present invention further provides fusion proteins comprising an APP protein, homologous polypeptide, or peptide fragment of the present invention joined to a carrier or fusion partner. The present invention further provides analogs and derivatives of an APP protein, homologous polypeptide, peptide fragment, or fusion protein of the present invention.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, Omp20, with or without signal sequence. In a preferred embodiment, the isolated Omp20-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:1 from about nt 329 to about nt 790. In a more preferred embodiment, the isolated Omp20-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:1 from about nt 272 to about nt 790.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, OmpW, with or without signal sequence. In a preferred embodiment, the isolated OmpW-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:3 from about nt 439 to about nt 1023. In a more preferred embodiment, the isolated OmpW-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:3 from about nt 376 to about nt 1023.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, Omp27, with or without signal sequence. In a preferred embodiment, the isolated Omp27-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:5 from about nt 238 to about nt 933. In a more preferred embodiment, the isolated Omp27-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:5 from about nt 157 to about nt 933.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, OmpA1, with or without signal sequence. In a preferred embodiment, the isolated OmpA1-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:7 from about nt 671 to about nt 1708. In a more preferred embodiment, the isolated OmpA1-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:7 from about nt 614 to about nt 1708.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, OmpA2, with or without signal sequence. In a preferred embodiment, the isolated OmpA2-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:9 from about nt 254 to about nt 1306. In a more preferred embodiment, the isolated OmpA2-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:9 from about nt 197 to about nt 1306.
The present invention further provides an isolated polynucleotide molecule that is homologous to any of the aforementioned polynucleotide molecules of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to any of the APP proteins of the present invention. The present invention further provides an isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned polynucleotide molecules of the present invention. In a non-limiting embodiment, the substantial portion of a polynucleotide molecule of the present invention encodes a peptide fragment of an APP protein or homologous polypeptide of the present invention. The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a fusion protein comprising an APP protein, homologous polypeptide, or peptide fragment of the present invention joined to a carrier or fusion partner.
The present invention further provides oligonucleotide molecules that are useful as primers for amplifying any of the polynucleotide molecules of the present invention, or as diagnostic reagents. Specific though non-limiting embodiments of such oligonucleotide molecules include oligonucleotide molecules having nucleotide sequences selected from the group consisting of any of SEQ ID NOS:15-47 and 49-93.
The present invention further provides compositions and methods for cloning and expressing any of the polynucleotide molecules of the present invention, including recombinant cloning vectors and recombinant expression vectors comprising a polynucleotide molecule of the present invention, host cells transformed with any of said vectors, and cell lines derived therefrom.
The present invention further provides a recombinantly-expressed APP protein, homologous polypeptide, peptide fragment, or fusion protein encoded by a polynucleotide molecule of the present invention.
The present invention further provides a vaccine for protecting swine against APP, comprising an immunologically effective amount of one or more antigens of the present invention selected from the group consisting of an APP protein, homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention capable of inducing, or contributing to the induction of, a protective response against APP in swine; and a veterinarily acceptable carrier or diluent. The vaccine of the present invention can further comprise an adjuvant or other immunomodulatory component. In a non-limiting embodiment, the vaccine of the present invention can be a combination vaccine for protecting swine against APP and, optionally, one or more other diseases or pathological conditions that can afflict swine, which combination vaccine has a first component comprising an immunologically effective amount of one or more antigens of the present invention selected from the group consisting of an APP protein, homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention capable of inducing, or contributing to the induction of, a protective response against APP in swine; a second component comprising an immunologically effective amount of a different antigen capable of inducing, or contributing to the induction of, a protective response against a disease or pathological condition that can afflict swine; and a veterinarily acceptable carrier or diluent.
The present invention further provides a method of preparing a vaccine that can protect swine against APP, comprising combining an immunologically effective amount of one or more antigens of the present invention selected from the group consisting of an APP protein, homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention capable of inducing, or contributing to the induction of, a protective response against APP in swine, with a veterinarily acceptable carrier or diluent, in a form suitable for administration to swine.
The present invention further provides a method of vaccinating swine against APP, comprising administering a vaccine of the present invention to a pig.
The present invention further provides a vaccine kit for vaccinating swine against APP, comprising a container comprising an immunologically effective amount of one or more antigens of the present invention selected from the group consisting of an APP protein, homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention capable of inducing, or contributing to the induction of, a protective response against APP in swine. The kit can further comprise a second container comprising a veterinarily acceptable carrier or diluent.
The present invention further provides antibodies that specifically bind to an APP protein of the present invention.
The present invention further provides diagnostic kits. In a non-limiting embodiment, the diagnostic kit comprises a first container comprising an APP protein, homologous polypeptide, peptide fragment, fusion protein, analog, or derivative of the present invention that will specifically bind to porcine antibodies directed against an APP protein; and a second container comprising a secondary antibody directed against the porcine anti-APP antibodies. The secondary antibody preferably comprises a detectable label. Such a diagnostic kit is useful to detect pigs that currently are, or have previously been, infected with APP, or that have seroconverted as a result of vaccination with a vaccine of the present invention. In a different non-limiting embodiment, the diagnostic kit comprises a first container comprising a primary antibody that binds to an APP protein of the present invention; and a second container comprising a secondary antibody that binds to a different epitope on the APP protein, or that binds to the primary antibody. The secondary antibody preferably comprises a detectable label. In a different non-limiting embodiment, the diagnostic kit comprises a container comprising a polynucleotide molecule or oligonucleotide molecule of the present invention that is useful to specifically amplify an APP-specific polynucleotide molecule of the present invention. These latter two diagnostic kits are useful to detect pigs that are currently infected with APP. 
FIGS. 1AandB. Western blot analysis of antibodies present in 1(a) serum, and 1(b) bronchial lymph node (BLN) tissue explant supernatants from pig No. 803 challenged with APP serotype-5 and heterologously rechallenged with APP serotype-7. All BLN tissue explant supernatants collected after 24 or 48 hr of incubation contained antibodies that specifically recognized APP proteins. The antibodies from the BLN tissue explant supernatants highlighted several low molecular weight proteins present in APP serotypes-1, 5, and 7.
FIG. 2. Western blot analysis of cross-reactivity of antibodies present in BLN tissue explant supernatants from pig No. 803 against whole bacterial cell antigens from each of the twelve different APP serotypes, demonstrating that at least three of the low molecular weight proteins recognized by antibodies induced by heterologous rechallenge were present in all twelve APP serotypes. Antibodies present in this particular BLN supernatant also recognized other protein bands.
FIG. 3. Western blot analysis demonstrating that reactivity of antibodies in BLN tissue explant supernatants from pig No. 803 against the low molecular weight proteins is restricted to proteins present in the cell pellets (cells) rather than bacterial cell supernatants and 4(b) serum from pig No. 803, against proteins purified from APP serotype-7 by continuous flow electrophoresis. Four protein bands with molecular weights of about 19-20, about 23, about 27, and about 29 kDa, respectively, were identified using this procedure.
FIG. 5. Alignment of deduced amino acid sequences of APP OmpA1 (SEQ ID NO:8) and APP OmpA2 (SEQ ID NO:10) proteins. The two proteins share 73.1% amino acid identity.
FIG. 6. Alignment of OmpW protein from Vibrio cholerae and OmpW (SEQ ID NO:4) protein from APP. The two proteins share 44.9% amino acid identity.